Method for improving flavor and flavonoid substance content of lightly fermented tea

Abstract

The invention discloses a method for improving flavor and flavonoid substance content of lightly fermented tea. Specific plant materials and tea leaves are jointly fermented through aspergillus niger and then loaded into small green oranges, the flavor of the obtained lightly-fermented tea is obviously improved, the lightly-fermented tea is obviously superior to other flower material fermented tea in liquor color, aroma and taste, the content of main aroma substances is also increased, the flower aroma is obviously improved, and the citrus aroma is weakened; after the specific plant materials and the tea leaves are fermented together, synthesis of part of flavonoid substances in the small green orange fermented tea can be promoted, the content of the flavonoid substances is increased, the content of narirutin, naringenin 7-O-glucoside and the like is remarkably increased, and the flavor of the small green orange tea is improved. Meanwhile, light fermentation is short in fermentation time and high in temperature, growth of other infectious microbes can be effectively inhibited, more time and energy cost are saved, simplicity, convenience, rapidness and easiness in implementation are achieved, and the problems of complexity, long time, unstable flavor and quality and the like of a traditional fermentation process are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and more specifically, relates to a method for improving the flavor and flavonoid content of lightly fermented tea. Background Art

[0002] Light fermented acid tea is produced by Aspergillus niger ( Aspergillus niger ) is made by high temperature and short-time fermentation. After fermentation, the free amino acid content of tea leaves increases significantly, the gallic acid content increases and it is rich in non-ester catechins. The tea soup is weakly acidic and has a significant sweet aftertaste. Although the flavor of the tea has been improved to a certain extent, there are still problems with a single flavor and lack of layering.

[0003] At present, methods for improving the flavor of tea include scenting, fermentation, baking, aging, and adding compound substances. The scenting process combines tea with floral and fruity fragrances, allowing the tea to fully absorb their aroma, forming a unique series of scented teas, which can bring a mellow taste and rich layers to Pu'er tea, black tea, etc., but the process is costly and complicated. Among them, it takes a long time to ferment tea leaves traditionally, and the growth of microorganisms is difficult to control; and due to different degrees of fire baking, the aroma of tea leaves is fixed and different fire fragrances are developed, but the degree of baking needs to be precisely controlled, which is prone to produce burnt smells; in addition, aging allows tea leaves to mature naturally in the precipitation of time and add flavor, but this method takes a long time; and the added compound substances such as cinnamon and tangerine peel bring new flavor dimensions to tea leaves, and it is also necessary to accurately control the proportion of their addition to mix and blend to create a variety of taste combinations, which requires a certain amount of time to explore and study, otherwise it will be counterproductive. Therefore, in order to further improve the problem of single flavor and lack of strong layers of lightly fermented tea, it is necessary to develop more new methods and products to enhance the flavor, aroma and flavonoid content of lightly fermented tea. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the problems of single flavor and insufficient layering of existing lightly fermented tea, and to provide a method for improving the flavor of lightly fermented tea.

[0005] The first object of the present invention is to provide a method for improving the flavor and flavonoid content of lightly fermented tea.

[0006] The second object of the present invention is to provide an application of the method for improving the flavor and flavonoid content of lightly fermented tea.

[0007] The third object of the present invention is to provide a kind of small green citrus lightly fermented tea.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a method for improving the flavor and flavonoid content of lightly fermented tea, comprising the following steps: S1. Mix tea leaves and plant materials in a mass ratio of (8-9):(1-2) and adjust the water content to 20-60%; then inoculate Aspergillus niger at a liquid-to-mass ratio of 1 mL:(90-110) g and ferment for 2-5 days to obtain light fermented tea; S2. After the fermentation is completed, the magnolia lightly fermented tea is mixed and filled into small green tangerines, dried, and vacuum-stored, which can enhance the flavor and flavonoid content of the small green tangerine-lightly fermented tea.

[0009] The present invention ferments specific plant materials and tea leaves by Aspergillus niger and then puts them into Xiaoqinggan, so that the flavor of Xiaoqinggan lightly fermented tea is significantly improved, the content of its main aroma substances is improved, the flower fragrance is significantly improved, the citrus fragrance is weakened, the smell of Xiaoqinggan can be well improved, the flower fragrance is strong, and the synthesis of flavonoids in Xiaoqinggan fermented tea can be promoted, and the content and activity of flavonoids in tea leaves can be increased; among them, the improvement effect of compounding magnolia, gardenia and osmanthus is the most obvious. In addition, the lightly fermented tea prepared by the method of the present invention greatly shortens the fermentation time, the method is simple and convenient, and it saves more time and energy costs, and is easy to implement. It solves the problems of single flavor of lightly fermented tea, long fermentation time of traditional Xiaoqinggan fermented tea, direct mixing of flower materials to fill Xiaoqinggan to affect the flavor and taste of tea, and the weakening of flower fragrance, unbalanced flavor, and low content of active substances in tea leaves in existing Xiaoqinggan flower tea.

[0010] Preferably, in step S1, the Aspergillus niger is an Aspergillus niger spore suspension.

[0011] Furthermore, the preparation method of the Aspergillus niger spore suspension is as follows: take the Aspergillus niger glycerol tube stored at -20°C, use an inoculation needle to inoculate it on a PDA plate, and culture it at 30°C for 5-7 days. After the surface of the culture medium is full of spores, the spores are washed with 0.1% Tween 80 sterile water and transferred to a centrifuge tube, and shaken evenly to form an Aspergillus niger spore suspension; use a hemocytometer to adjust the concentration of the spore suspension.

[0012] More preferably, the concentration of the Aspergillus niger spore suspension is not less than 1-5×10 8 Pieces / mL.

[0013] Preferably, the water content in step S1 is 40-50%.

[0014] Preferably, the fermentation temperature in step S1 is 30-55°C.

[0015] Preferably, the drying temperature in step S2 is 80-100°C.

[0016] Preferably, the Aspergillus niger is Aspergillus niger RAF106 strain, which has been deposited in China General Microbiological Culture Collection Center on August 27, 2014, with a deposit number of CGMCC NO.9608.

[0017] More preferably, the magnolia flowers are dried magnolia flowers; dried magnolia flowers can be preserved for a long time, thereby ensuring that they remain in a stable state during the fermentation process, and the dried flowers are more convenient to handle and do not require frequent replacement or processing, thereby reducing the trouble during the fermentation process.

[0018] The present invention provides application of the method in promoting the synthesis of flavonoids in Xiaoqinggan tea or in increasing the content of flavonoids in Xiaoqinggan tea.

[0019] Furthermore, the flavonoid substances include flavonoids, isoflavones, neoflavonoids, total flavonoids, monomeric flavonoids, polymeric flavonoids, etc.

[0020] The present invention provides application of the method in preparing lightly fermented tea with strong floral fragrance and high flavonoid content.

[0021] The invention also provides a small green citrus lightly fermented tea, which is prepared by the method.

[0022] As a more specific implementation scheme, the present invention provides a method for preparing lightly fermented citrus tea: 90 g of Yunnan large-leaf tea and 10 g of plant flowers are weighed separately into a fermentation bag, and the tea leaves are rehydrated with sterile water to a water content of 45%. A spore suspension of Aspergillus niger (with a concentration of 1 to 5×10 8 / mL) are evenly inoculated on the tea leaves. The inoculated tea leaves are placed in a 42°C fermentation incubator for fermentation. During the fermentation period, the tea leaves are turned over every day and fermented for 4 days. After the fermentation is completed, the tea leaves are mixed evenly and stuffed into the small green citrus, placed in an 80°C oven for drying, and vacuum packaged for storage to obtain the small green citrus-magnolia lightly fermented tea.

[0023] In particular, the present invention shows that the bitter taste of fermented tea can be improved by using small green citrus, magnolia, gardenia, and osmanthus, which will not cover the aroma of tea, but also increase the aroma. Based on this, gardenia and osmanthus are used in the preparation of the above-mentioned small green citrus light fermented tea. Those skilled in the art can expect that the small green citrus light fermented tea prepared by this method can also achieve the effect of improving the flavor of light fermented tea and the content of flavonoids.

[0024] The present invention has the following beneficial effects: The invention provides a method for improving the flavor and flavonoid content of lightly fermented tea. After a specific plant material and tea leaves are co-fermented by Aspergillus niger, they are loaded into a small green citrus fruit. The flavor of the obtained small green citrus fruit lightly fermented tea is significantly improved, the content of its main aroma substances is improved, the flower fragrance is strong, and the citrus fragrance is weakened; the specific plant material and tea leaves are co-fermented, and the synthesis of some flavonoids in the small green citrus fruit fermented tea can be promoted, the content of flavonoids can be increased, the content of naringin and naringenin 7-O-glucoside can be significantly increased, the flavor of the small green citrus fruit tea can be improved, and the anti-inflammatory, antioxidant and anti-cancer activities of the small green citrus fruit tea can be further enhanced. The sensory scores of the small green citrus fruit lightly fermented tea prepared by the method of the invention are improved in terms of soup color, aroma and taste.

[0025] At the same time, the method provided by the present invention can improve the flavor of Xiaoqinggan tea, enhance the floral fragrance, and increase the content of effective substance flavonoids. The fermentation method is simple and fast, the fermentation time is short, requiring 2 to 5 days, and the fermentation temperature is high, reaching above 45°C, which can effectively inhibit the growth of other bacteria, save time and energy costs, is easy to implement, has low labor costs, and can also avoid the problems of cumbersome traditional fermentation processes, long time, unstable flavor and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effects of different compounds on total polyphenols in fermented tea.

[0027] Figure 2 The effect of different compound substances on total flavonoids in fermented tea.

[0028] Figure 3 It is the tea base and tea soup of different compound fermented teas.

[0029] Figure 4 The effects of different compound fermented teas on aroma ((a) PCA chart and (b) radar chart).

[0030] Figure 5 This is the aroma radar chart of different compound lightly fermented teas.

[0031] Figure 6 This is a graph showing the effects of different flowers on Xiaoqinggan tea leaves, tea base and tea soup after fermentation.

[0032] Figure 7 The overall profile of aroma components in groups X1 and X2 (in the figure, (a) the number of aroma components of different categories; (b) the proportion of aroma components of different categories; (c) heat map); X1 is Xiaoqinggan-fermented tea; X2 is Xiaoqinggan-Yulan fermented tea).

[0033] Figure 8The OPLS-DA model analysis results of aroma components in X1 and X group 2 (in the figure (a) score graph; (b) cross-validation graph; X1 is Xiaoqinggan-fermented tea; X2 is Xiaoqinggan-Yulan fermented tea).

[0034] Fig. 9 It is the total ion current diagram of volatile substances in the samples (in the figure, (a) total ion current diagram of volatile substances in sample X1 and (b) total ion current diagram of volatile substances in sample X2; X1 is fermented tea of ​​citrus aurantium and X2 is fermented tea of ​​citrus aurantium and magnolia).

[0035] Fig.10 The overall profile of flavonoids in groups X1 and X2 (in the figure (a) Venn diagram and (b) heat map; X1 is Xiaoqinggan-fermented tea; X2 is Xiaoqinggan-Yulan fermented tea).

[0036] Fig.11 The results of differential metabolite analysis of flavonoids in groups X1 and X2 ((a) OPLS-DA score graph; (b) model cross-validation graph; (c) volcano graph; (d) violin graph; X1 is citrus aurantium-fermented tea; X2 is citrus aurantium-magnolia fermented tea).

[0037] Fig.12 Schematic diagram of the structures of the main differential metabolites between groups X1 and X2. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Aspergillus niger ( Aspergillus niger ) The RAF106 strain is the preliminary research result of the applicant's research team. The preservation date of the RAF106 strain is August 27, 2014, and the preservation number is CGMCCNO.9608. The preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); the address of the preservation unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, and it is recorded in the existing patent document: CN 10467384 A.

[0041] Preparation of Aspergillus niger spore suspension: Take the glycerol tube of Aspergillus niger RAF106 strain stored at -20°C, use an inoculation needle to inoculate it on a PDA plate, and culture it at 30°C for 5-7 days. After the surface of the culture medium is full of spores, wash the spores with 0.1% Tween 80 sterile water and transfer them to a 10 mL centrifuge tube, and shake evenly to form an Aspergillus niger spore suspension. Use a hemocytometer to adjust the concentration of the spore suspension to 1~5×10 8 Pieces / mL.

[0042] The source information of tea leaves and flower materials used in the following examples is shown in Table 1.

[0043] Table 1 Main raw materials and their sources

[0044] Example 1 Effects of different flowers on tea flavor 1. Preparation of lightly fermented tea Weigh 90 g of Yunnan large-leaf tea and 10 g of different materials (nasturtium, peony, rose (Yunnan), osmanthus, magnolia, jasmine, gardenia, rose, honeysuckle, chrysanthemum, 2022 new mandarin orange, grapefruit peel, green mandarin orange, small green mandarin orange, loquat leaf, mulberry leaf, hawthorn leaf, mint, and nepeta spicate) in a fermentation bag, and rehydrate the tea leaves with sterile water to a water content of 45%. Add Aspergillus niger spore suspension (concentration of 1-5×10 8 The inoculated tea leaves were placed in a 42°C fermentation incubator for fermentation. The tea leaves were turned over every day during the fermentation period and fermented for 4 days. After the fermentation, lightly fermented teas fermented with different materials were obtained.

[0045] 2. Determination method of total polyphenol content Total polyphenols are one of the main secondary metabolites of plants and an important indicator for evaluating tea quality. Tea polyphenols are the main component of the bitter taste of tea soup. Referring to GBT8313-2018 "Test Method for Tea Polyphenols and Catechins in Tea", the total polyphenol content in tea was detected by the Folin phenol method. 0.2 g of tea powder was mixed with 5 mL of 70% methanol and heated in a 70°C water bath for 10 min. Subsequently, the mixture was centrifuged at 3500 r / min for 10 min, and the process was repeated twice. The supernatant obtained twice was the tea polyphenol extract. 1 mL of 100-fold diluted tea polyphenol extract was mixed with 5 mL of 10% Folin phenol and reacted for 5 minutes, then 4 mL of 7.5% Na2CO3 solution was added, shaken thoroughly, and allowed to stand at room temperature for 1 hour, and then the absorbance was measured at a wavelength of 765 nm using a 96-well plate. According to the standard curve of total polyphenols (y = 0.0083 x + 0.0706, R 2= 0.9981) was used for quantification. The fermented tea group without flower materials was taken as the control group (FT).

[0046] The total polyphenol content of co-fermented teas from different materials was determined. Figure 1 As shown, the total polyphenol content of Trollius co-fermented tea was significantly increased compared with FT ( p <0.05), there was no difference in the total polyphenol content of peony flower, Qianfengxi berry tea, loquat leaf and mulberry leaf co-fermented tea, while other flowers and citrus substances significantly reduced the total polyphenol content of co-fermented tea ( p <0.05). This indicates that co-fermentation with citrus fruits can reduce the bitterness of tea.

[0047] 3. Determination method of total flavonoids Flavonoids are widely present in plants in nature. They are secondary metabolites of plants and are an important class of polyphenolic compounds that affect the taste and color of tea. They are also important astringent compounds in tea. The specific method for determining the total flavonoid content of co-fermented tea is as follows: (1) Test solution preparation: Weigh 1 mg of rutin standard into a 1.5 mL centrifuge tube and dissolve it in 1 mL of methanol to prepare a 1 mg / mL rutin standard solution. Pipette 100 μL of rutin standard solution into a 1.5 mL centrifuge tube containing 900 μL of 60% anhydrous ethanol to prepare a 100 μg / mL standard test solution. Prepare 80, 40, 20, 10, and 5 μg / mL standard test solutions in sequence.

[0048] (2) Preparation of sample extract: Weigh 0.10 g of ground tea sample into a 10 mL centrifuge tube, add 5 mL of 60% ethanol, perform ultrasonic-assisted extraction for 30 min, centrifuge at 8000 r / m for 5 min, remove the supernatant, repeat the extraction once, and combine the supernatants in a 10 mL volumetric flask to make up to volume.

[0049] (3) Determination: Pipette 100 μL of standard test solution and sample solution into 1.5 mL centrifuge tubes, add 150 μL of 5% AlCl3 solution, then add 100 μL of acetic acid-sodium acetate buffer (pH = 5.5), dilute to 1.0 mL with 60% ethanol, let stand for 30 min, measure the absorbance of the extract at 400 nm, and use 60% ethanol test solution as blank control. According to the absorbance values ​​of each standard test solution, establish a flavonoid concentration-absorbance value standard curve (y = 0.0021 x + 0.0435, R 2 = 0.9975), the concentration of the sample flavonoid compounds was obtained according to the standard curve, and the content of the flavonoid compounds in the tea sample was calculated. The calculation formula is as follows:

[0050] Where: X: total flavonoids content in tea (mg / g); x1: Calculate the total flavonoids concentration (μg / mL) in the test solution according to the standard curve; N: dilution factor of the assay solution; V: volume of tea extract (mL); M: weighed mass of tea sample (g).

[0051] The total flavonoid content of co-fermented teas from different materials was determined. Figure 2 As shown in Figure 2, compared with FT, the co-fermentation of nasturtium, peony flower, Yunnan rose, magnolia, loquat leaf and hawthorn leaf significantly increased the total flavonoid content ( p <0.05). However, most flower and citrus substances will significantly reduce the total flavonoids in co-fermented tea ( p <0.05). This indicates that co-fermentation with citrus fruits can further reduce the astringency of tea.

[0052] 3. Sensory evaluation Subsequently, the 20 co-fermented teas were subjected to sensory evaluation. The sensory evaluation method for tea was based on the national standard GB / T23776-2018 "Tea Sensory Evaluation Method" for yellow tea sensory evaluation. The sensory evaluation team consisted of at least five people with sensory experience. The sensory evaluation was based on three factors: soup color, aroma, and taste. The evaluation process involved adding 150 mL of 85°C water to 3 g of sample and brewing for 3 minutes. Subsequently, the color, aroma, and taste were immediately evaluated. According to the scoring criteria as shown in Table 2, the total score calculation formula is as follows: Total score = 20% × soup color score + 30% × aroma score + 50% × taste score Table 2 Tea sensory evaluation table

[0053] The sensory score table of different compound fermented teas is shown in Table 3. Compared with FT (81.25 ± 13.04 points), the compounds with higher total sensory scores include small green citrus (89.8 ± 1.15 points), magnolia (89.48 ± 8.71 points), gardenia (88.6 ± 5.51 points) and osmanthus (87.94 ± 3.2 points). The sensory listeners can feel a more pleasant fruity or floral aroma, and the taste is smooth with obvious sweetness, which may be related to the significant decrease in their bitter substances (total flavonoids and total polyphenols). For substances with strong odors (such as chrysanthemums) or medicinal and edible substances (such as golden lotus, honeysuckle, and schizonepeta spicate), the sensory scores are generally low. Their own odors are too strong, so that they will cover the taste of the tea itself, making the sensory listeners feel bad. In addition, the tea leaves, tea bases and tea soups of different co-fermented teas have their own characteristics, such as Figure 3 shown.

[0054]

[0055] 4. Electronic nose measurement The electronic nose was used to measure the aroma of teas with higher sensory scores (small green orange, magnolia, gardenia and osmanthus) and FT. The specific method of electronic nose detection (using odor scanner) was as follows: 1.0 g of tea samples were weighed and placed in 50 mL conical flasks, sealed with sealing film, and left to stand for 30 min. The electronic nose aroma detection head was inserted into the conical flask to collect aroma data. Detection conditions: injection flow rate of 300 mL / min, cleaning channel of 120 s, automatic zeroing of 10 s, sample preparation time of 5 s, measurement time of 110 s, and response signal interval of 106~108 s for data analysis. The performance description of the 10 sensors of the electronic nose used is shown in Table 4.

[0056] Table 4 Sensors used in PEN3 and their performance description

[0057] In the PCA graph, if Figure 4 As shown in a, the contribution rate of the first principal component is 98.17%, the contribution rate of the second principal component is 1.58%, and the total contribution rate is 99.75%, indicating that the two principal components can represent the main information of the aroma of tea leaves at different fermentation times. The five samples are separated from each other, indicating that the aroma of the five samples has certain differences, among which the points of Xiaoqinggan and FT are the farthest apart, indicating that Xiaoqinggan has the best effect on enhancing the aroma of FT.

[0058] The radar chart can intuitively reflect the response of the 10 sensors of the electronic nose to the aroma of compound fermented tea, such as Figure 4 As shown in Figure b, the signals of the composite fermented tea to W1W (sensitive to sulfides), W5S (high sensitivity, very sensitive to nitrogen oxides) and W2W (aromatic components, sensitive to organic sulfides) are relatively strong. Compared with FT, the response values ​​of the co-fermented teas of citrus aurantium, magnolia, gardenia and osmanthus to the W1W, W5S and W2W sensors are stronger, among which the response value of citrus aurantium is the strongest. The tea aroma response radar is consistent with the PCA analysis results.

[0059] In summary, the results show that the use of small green citrus, magnolia, gardenia, and osmanthus can better improve the bitter taste of fermented tea, will not cover the aroma of the tea, and can also increase the aroma.

[0060] Example 2 Preparation of Xiaoqinggan-Yulan Lightly Fermented Tea Weigh 90 g of Yunnan large-leaf tea and 10 g of magnolia (dried flower material) into a fermentation bag, and rehydrate the tea leaves with sterile water to a moisture content of 45%. Add Aspergillus niger spore suspension (concentration of 1-5×108 / mL) are evenly inoculated on the tea leaves. The inoculated tea leaves are placed in a 42°C fermentation incubator for fermentation. During the fermentation period, the tea leaves are turned over every day and fermented for 4 days. After the fermentation is completed, the tea leaves are mixed evenly and stuffed into the small green citrus, placed in an 80°C oven for drying, and vacuum packaged for storage to obtain the small green citrus-magnolia lightly fermented tea.

[0061] Example 3 Preparation of Xiaoqinggan-Yulan Lightly Fermented Tea Weigh 80 g of Yunnan large-leaf tea and 20 g of magnolia in a fermentation bag, and rehydrate the tea with sterile water to a water content of 60%. Add Aspergillus niger spore suspension (concentration of 1-5×10 8 / mL) are evenly inoculated on the tea leaves. The inoculated tea leaves are placed in a 55°C fermentation incubator for fermentation. During the fermentation period, the tea leaves are turned over every day and fermented for 5 days. After the fermentation is completed, the tea leaves are mixed evenly and stuffed into the small green citrus, placed in a 90°C oven for drying, and vacuum packaged for storage to obtain the small green citrus-magnolia lightly fermented tea.

[0062] Example 4 Preparation of Xiaoqinggan-Yulan Lightly Fermented Tea Weigh 85 g of Yunnan large-leaf tea and 15 g of magnolia in a fermentation bag, and rehydrate the tea with sterile water to a moisture content of 20%. Add Aspergillus niger spore suspension (concentration of 1-5×10 8 / mL) are evenly inoculated on the tea leaves. The inoculated tea leaves are placed in a 30°C fermentation incubator for fermentation. During the fermentation period, the tea leaves are turned over every day and fermented for 2 days. After the fermentation is completed, the tea leaves are mixed evenly and stuffed into the small green tangerines, placed in a 100°C oven for drying, and vacuum packaged for storage to obtain the small green tangerine-magnolia lightly fermented tea.

[0063] Example 5: Small green citrus-osmanthus lightly fermented tea The preparation of the fermented tea in this example is the same as that in Example 2, with the only difference being that the magnolia flower is replaced by osmanthus flower.

[0064] Example 6 Small green citrus-gardenia lightly fermented tea The preparation of the fermented tea in this example is the same as that in Example 2, with the only difference being that the magnolia flower is replaced by the gardenia flower.

[0065] Comparative Example 1 Preparation of Xiaoqinggan-Lightly Fermented Tea Weigh 100 g of Yunnan large-leaf tea, rehydrate the tea with sterile water to a water content of 45%, and add Aspergillus niger spore suspension (concentration of 1-5×10 8 / mL) were evenly inoculated on the tea leaves, and after the tea leaves were mixed evenly, they were placed in a 42°C fermentation incubator for fermentation. During the fermentation period, the tea leaves were turned over every day and fermented for 4 days. After the fermentation was completed, the tea leaves were mixed evenly and stuffed into the small green tangerines and placed in an 80°C oven for drying, and vacuum packed for storage to obtain the small green tangerine-lightly fermented tea.

[0066] Comparative Example 2 Magnolia-Small Green Orange Tea The preparation of the small green citrus tea in this comparative example is the same as that in comparative example 1, the only difference is that when filling the tea leaves, the magnolia flowers and the fermented tea leaves are directly mixed in a mass ratio of 9:1 and then stuffed into the small green citrus and dried.

[0067] Test Example 1 Flavor of Xiaoqinggan Lightly Fermented Tea Based on the research of Example 1, the fermented tea prepared in Examples 2, 5-6 and Comparative Example 1 were compared, and sensory analysis and electronic nose measurement were performed in the same manner as in Example 1.

[0068] The aroma radar charts of different compound lightly fermented teas are as follows: Figure 5 As shown in the figure, the signals of W1W (sensitive to sulfides), W5S (high sensitivity, very sensitive to nitrogen oxides) and W2W (aromatic components, sensitive to organic sulfides) of xiaoqinggan-magnolia, xiaoqinggan, xiaoqinggan-gardenia, and xiaoqinggan-osmanthus are enhanced in turn. Among them, the combination of magnolia lightly fermented tea and xiaoqinggan has the best effect, which can significantly enhance the aroma and flavor of tea.

[0069] The results of sensory evaluation are shown in Table 5. The soup colors of different combinations of small green citrus and flowers are as follows: Figure 6 As shown, the combination of osmanthus fragrans and magnolia had the highest sensory scores for soup color, aroma and taste, with a total score of 90.28 ± 4.43 points, which is consistent with the results of the electronic nose. This shows that the combination of lightly fermented teas made of osmanthus, gardenia and magnolia with osmanthus fragrans can greatly enhance the flavor of fermented tea.

[0070] Table 5 Effects of different flowers and small green citrus on sensory perception

[0071] Test Example 2 Effect of Magnolia on Volatile Substances in Xiaoqinggan-Lightly Fermented Tea The aroma components and their contents in the Xiaoqinggan lightly fermented tea (referred to as Group X1) of Comparative Example 1 and the Xiaoqinggan-Yulan lightly fermented tea (referred to as Group X2) of Example 2 were qualitatively and quantitatively analyzed by HS-SPME-GC-MS technology. The specific method is: Preparation of internal standard solution: Take 0.01 mL of ethyl decanoate standard stock solution in a 100 mL volumetric flask, make up to volume with anhydrous ethanol to prepare a standard solution with a mass concentration of 8.71 g / L, and place it in aliquots at 4°C for later use.

[0072] Headspace solid phase microextraction conditions: Weigh 1 g of tea powder sample and place it in a 15 mL headspace bottle, add 0.5 g of sodium chloride and 5 mL of boiling deionized water, then add 10 μL of ethyl decanoate (8.71 mg / L), cover the bottle tightly, and shake well. Seal the bottle and place it in a 90°C water bath for 10 min, insert the 30 / 50 μm DVB / CAR / PDMS extraction head into the extraction bottle for headspace extraction for 30 min, insert it into the gas chromatography injection port for decomposition at 250°C for 5 min, and perform GC-MS / MS analysis.

[0073] Gas chromatography conditions: injection port temperature 250 °C, chromatographic column TG-5MS (60 m×0.25 mm, 0.5 μm); injection mode: non-split; carrier gas is He gas, flow rate 1.2 mL / min; temperature program: 60 °C for 1 min, increase to 150 °C at 3 °C / min, maintain for 2.0 min; increase to 180 °C at 3 °C / min, maintain for 3.0 min; increase to 240 °C at 4 °C / min, maintain for 2.0 min.

[0074] Mass spectrometry conditions: ion source temperature was 230 °C, interface temperature was set to 230 °C, and scan was performed in the range of 35-600 m / z.

[0075] Qualitative and quantitative analysis: The unknown compounds are qualitatively identified by matching the Willey / Nist spectral library, and the content of the aroma components is obtained by comparing the peak area with that of the internal standard. The formula is as follows:

[0076] Where: C: aroma component content (μg / L); C0: internal standard concentration (8.71 μg / L); N: aroma component peak area; M: peak area of ​​internal standard substance.

[0077] The results of the test are as follows Figure 7 As shown in a, 68 and 70 aroma components were detected in X1 and X2, respectively, and the total contents of aroma components were X1 (1994.87 μg / L) and X2 (1237.09 μg / L), respectively. Although the number of substance categories increased by 2 after adding the co-fermented magnolia flowers, the concentration of the substances showed a downward trend, indicating that magnolia flowers can significantly improve the aroma of small green oranges.

[0078] Since there are some differences in the number and content of aroma components between X1 and X2, further analysis shows that alcohols, esters and alkenes are the main categories of the two ( Figure 7b). It shows that the proportion of alcohols in X2 (30%) is greater than that in X1 (27%); the proportion of esters is not much different, X1 (27%) and X2 (26%) respectively; the proportion of hydrocarbons is also not much different, X1 (21%) and X2 (22%) respectively, indicating that the overall fragrance of X1 and X2 has fruity and sweet aromas, but X2 has a stronger floral aroma.

[0079] in addition, Figure 7 c shows that there are significant differences in the content of aroma components between X1 and X2. The specific statistical results of the main aroma components are shown in Table 6, which shows that the main aroma components in the two types of Xiaoqinggan tea are significantly different. Among them, 2-(methylamino)benzoic acid methyl ester (488.34 ± 211.91 μg / L), (+)-limonene (245.56 ± 13.38 μg / L) and carvacrol (115.41 ± 51.32 μg / L) are higher in X1 and are its main aroma components, showing a strong citrus aroma; while in X2, their content is reduced, weakening the citrus aroma.

[0080] Table 6 Statistical results of main aroma components

[0081] Test Example 3 Analysis of Differential Metabolites The aroma activity value (OAV) and orthogonal partial least squares discriminant analysis (OPLS-DA) of samples X1 and X2 were further analyzed to study the effect of magnolia on the aroma of small green citrus-lightly fermented tea.

[0082] Calculation of aroma activity value (OAV): OAV is the ratio of the concentration of a certain aroma component to its olfactory threshold. The threshold is obtained from relevant literature, and then OAV is calculated based on the quantitative results (Wang Mengqi et al., 2019), according to the following formula:

[0083] Where: OAV: activity value of aroma component; C: content of aroma components (μg / L); OT: threshold value of aroma components (μg / L).

[0084] The OAV analysis results are shown in Table 7. It can be seen that there are 9 substances with OAV greater than 0.1 (OAV value greater than 0.1 means that the concentration of aroma components exceeds the human olfactory threshold and can be perceived by humans) and 6 substances with OAV greater than 1 in the two samples. In the OPLS-DA model statistics, the parameter is R 2Y = 0.994 and Q 2 = 0.968. The closer the parameter values ​​are to 1, the better the applicability and predictability of the model. Figure 8 a), screen out substances with VIP values ​​greater than 1, and further combine them with t-test ( p <0.05) to screen for differential metabolites.

[0085] Table 7 Analysis of aroma activity values ​​and aroma description of aroma components of X1 and X2

[0086] Note: The OAV values ​​in the table are the average values ​​of samples in each group, a: aroma and threshold, derived from the literature (Hong Xin, 2022; Li Yongdi, 2019; Tang Zhongsheng, 2019).

[0087] Based on VIP>1, p <0.05, OAV>1, two different aroma compounds were screened between X1 and X2, namely (+)-limonene and trans-α,α-5-trimethyl-5-vinyltetrahydro-2-furanmethanol. The content of (+)-limonene in X2 (111.76± 11.4 μg / L) was 0.54 times lower than that in X1 (245.56± 13.4 μg / L), but the OAV reached 93.14, which contributed to the fruity aroma of tea. The content of trans-α,α-5-trimethyl-5-vinyltetrahydro-2-furanmethanol (floral aroma) in X2 was 12.08± 4.99 μg / L, and the OAV value was as high as 201.33. It was not detected in X1 and was a characteristic substance of X2 ( Fig. 9 ). This shows that after adding magnolia lightly fermented tea, Xiaoqinggan tea can not only maintain the original citrus fragrance, but also produce a rich floral fragrance.

[0088] In addition, in Comparative Example 2, directly mixing magnolia flowers and tea leaves and loading them into small green tangerines can also make the small green tangerine tea have a floral aroma, but since the volatile substance in the magnolia flowers is mainly linalool, which is highly volatile, its aroma weakens quickly; and after the magnolia flowers and tea leaves are fermented by Aspergillus niger, the content of trans-α,α-5-trimethyl-5-vinyltetrahydro-2-furanmethanol increases significantly, and after fermentation, linalool will be converted into linalool oxide: trans-α,α-5-trimethyl-5-vinyltetrahydro-2-furanmethanol, the threshold of this oxide is relatively low, so the fragrance of the magnolia flowers after fermentation will be more obvious and rich.

[0089] Test Example 4 Analysis of Differential Metabolites of Flavonoids in Fermented Tea LC-MS / MS was used to quantitatively analyze the active ingredients of flavonoids in fermented tea leaves in groups X1 and X2, and the effect of co-fermentation of magnolia and tea leaves on the flavonoids rich in citrus-lightly fermented tea was studied.

[0090] Test results such as Fig.10 As shown, there are 62 flavonoids detected in the two groups, of which 59 are common to X1 and X2, 2 are unique to X1, and 1 is unique to X2, indicating that there is little difference in the types of flavonoids between the X1 and X2 groups ( Fig.10 a). However, the clustering results showed that the concentration of flavonoids between X1 and X2 was quite different ( Fig.10 b).

[0091] To further explore the differences in flavonoids between groups X1 and X2, an OPLS-DA model was constructed. The statistical parameters of the model were R2Y = 0.997 and Q2 = 0.886. The model was verified to have good applicability and predictability ( Fig.11 ab), can be further used for screening of differential metabolites.

[0092] After OPLS-DA analysis, substances with VIP values ​​greater than 1 were screened out, and their change trends were further determined by combining the FC values. p <0.05, |Log2FC|>1, 5 differential metabolites were obtained between X1 and X2. Compared with X1, the content of 4 substances in X2 increased and the content of 1 substance decreased. Among these differential metabolites, naringenin, naringenin 7-O-glucoside and mangiferin accounted for the majority in terms of content ( Fig.12 ). They accounted for 99.99% and 99.09% of the total differential metabolite contents of X1 and X2, respectively. The contents of some differential flavonoids are shown in Table 8.

[0093]

[0094]

[0095] Note: a, b in the table represent letters indicating significant differences. If two groups of letters are the same, it means there is no significant difference.

[0096] Among them, the content of naringenin in X2 (20.99 ± 2.59 nmol / g) increased by 1.12 times compared with X1 (9.88 ± 1.45nmol / g). Naringenin has antioxidant and anti-inflammatory activities. It also has anti-cancer, neuroprotective, stress-relieving, liver-protective, anti-allergic, anti-obesity and immunomodulatory effects. The content of naringenin 7-O-glucoside in X2 (15.05 ± 0.85 nmol / g) increased by 1.11 times compared with X1 (7.13 ± 0.35 nmol / g). It can play an anti-cancer and anti-diabetic role by activating the PI3K / Akt pathway and PPARγ. The content of mangiferin in X1 was 0.5 ± 0.12 nmol / g, which was not detected in X2, indicating that co-fermentation with magnolia flowers can promote the synthesis of some flavonoids in fermented citrus aurantium tea, increase its flavonoid content, and further enhance the anti-inflammatory, antioxidant and anti-cancer activities of citrus aurantium tea.

[0097] In summary, the co-fermentation study based on different flower-leaf citrus materials shows that the bitter taste of tea can be improved after the flower-leaf citrus is fermented with tea leaves. Although the flower materials are directly mixed in the small green citrus tea (Comparative Example 2), the small green citrus has a floral fragrance, but the direct mixing of the flower materials affects the flavor and taste of the tea, and its floral fragrance will still be weakened, making it difficult to balance the flavor and taste. The present invention has obtained that the specific flower materials are co-fermented with light fermented acid tea and then filled into the small green citrus, which can not only well improve the smell of the small green citrus, and its floral fragrance is rich, but also promote the synthesis of some flavonoids in the fermented tea of ​​the small green citrus, and increase the content and activity of flavonoids in the tea leaves; and the light fermentation preparation is simple and convenient, which can greatly shorten the fermentation time, save more time and energy costs, and can further increase the content of flavonoids while improving the flavor of the light fermented acid tea, and it is easier to achieve.

[0098] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0099] The references cited in this article are: Hong Xin. Study on characteristic aroma components and core functional microbial communities of different aroma types of Liubao tea[D]. Hunan Agricultural University, 2022.DOI:10.27136 / d.cnki.ghunu.2022.000344. Li Yongdi. Study on characteristic aroma components during the processing of Fuzhuan tea[D]. Hunan Agricultural University, 2019.DOI:10.27136 / d.cnki.ghunu.2019.000650. Tang Zhongsheng. Research on the change law of aroma components and characteristic aroma during the processing of litchi wine[D]. South China University of Technology, 2019.DOI:10.27151 / d.cnki.ghnlu.2019.004175.

Claims

1. A method for improving the flavor and flavonoids of light fermented sour tea, characterized in that: The following steps are involved: S1. Compound tea leaves and plant materials in a mass ratio of (8-9):(1-2) to adjust the water content to 20-60%; then inoculate Aspergillus niger at a liquid-to-mass ratio of 1 mL:(90-110) g, and ferment for 2-5 days to obtain light fermented tea; The plant material is selected from one or more of osmanthus, magnolia, and gardenia; S2. After fermentation, mix the lightly fermented tea with flowers and fill it into small green oranges, dry it, and store it in a vacuum, which can enhance the flavor of the lightly fermented tea.

2. The method according to claim 1, characterized in that: In step S1, the Aspergillus niger uses an Aspergillus niger spore suspension.

3. The method according to claim 2, characterized in that: The concentration of the Aspergillus niger spore suspension is not less than 1-5×10 8 Pieces / mL.

4. The method according to claim 1, characterized in that: The water content in step S1 is 40-50%.

5. The method according to claim 1, characterized in that: The fermentation temperature in step S1 is 30-55°C.

6. The method according to claim 1, characterized in that: The drying temperature in step S2 is 80-100°C.

7. The method according to claim 1, characterized in that: The Aspergillus niger adopts Aspergillus niger RAF106 strain, which has been deposited in China General Microbiological Culture Collection Center on August 27, 2014, with the deposit number of CGMCCNO.9608.

8. Use of the method according to any one of claims 1 to 7 in promoting the synthesis of flavonoids in Xiaoqinggan tea, or in increasing the content of flavonoids in Xiaoqinggan tea.

9. Use of the method according to any one of claims 1 to 7 in preparing citrus aurantium tea with strong floral aroma and high flavonoid content.

10. A lightly fermented tea of ​​small green citrus, characterized in that: Prepared by the method according to any one of claims 1 to 7.

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